小鼠肾小管上皮细胞的原代培养及生物学特性

目的探讨适用于小鼠肾小管上皮细胞(mRTECs)分离培养及鉴定的技术方法。方法采用机械研磨肾脏组织分离肾小管节段并结合Ⅱ型胶原酶消化分离培养法进行mRTECs原代培养,利用倒置显微镜观察细胞生长情况;锥虫蓝法、绘制生长曲线法、3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑蓝(MTT)法、DNA周期检测法分别测定和观察mRTECs传代成活率、生长情况及增殖特征;免疫荧光染色法鉴定细胞。结果获得的细胞3 d后从贴壁的肾小管节段边缘长出,7 d后呈铺路石样,生长迅速可传代;传代细胞成活率达96%以上;第1代(P1)、第3代(P3)细胞生长曲线近似"S"形,MTT法显示P1、P3细胞生长至第3～5天光密度值变化较明显;随传代次数的增加,第5代细胞生长曲线近似平缓,光密度值无明显变化,细胞逐渐衰老;P1细胞G0/G1和(S+G2)/M分别为78.9%和21.1%,P3细胞G0/G1和(S+G2)/M分别为82.1%和17.9%;P3细胞行免疫荧光染色细胞角质蛋白-18及上皮型钙黏素显示细胞质内蛋白表达阳性,细胞阳性率均达98%。结论机械研磨并结合Ⅱ型胶原酶消化培养法能成功培养mRTECs,建立稳定、高效的细胞分离和培养技术方法,为肾脏疾病与心血管功能相关性研究提供理想的细胞来源。

Primary culture and biological characteristics of renal tubular epithelial cells in mice

Objective To investigate effective methods to isolate,cultivate and identify the renal tubular epithelial cells(RTECs) in mice.Methods The renal tissue in mice was separated into tubular segments through mechanical grinding,which was cultivated by type Ⅱ collagen digestion.The cells growth state was observed by inverted microscope.Trypan blue staining,cell growth curves,3-(4,5-dimethyl-2-thiahiazo)-2,5-diphenyltetrazolium bromide(MTT) method and DNA cycle detection method were used to detect the passage survival rate,growth state and proliferation characteristic.The mRTECs biological characteristics was identified by immunofluorescence.Results The cells became confluent after 3 days and formed the cobblestones shape to be ready for passage after 7 days.The viability of the cells after the passage was more than 96% in trypan blue staining.The first(P1) and the third(P3) generation presented an "S" shape of curve of growth.MTT analysis showed that the cellular proliferation between P1 and P3 generation were quick and began to slow down after the fifth passage.After the immunofluoresence staining,the change of optical density value of MTT assay was obvious from the 3rd day to the 5th day.The ratio of G0/G1 phase and(S+G2)/M phase were 78.9% and 21.1% respectively at P1;82.1% and 17.9% respectively at P3.The expression of cytokeratin-18 and E-cadherin of mRTECs in immunofluorescence staining were both 98% positive.Conclusion Mechanical grinding method and enzyme digestion method are effective and stable ways to isolate and cultivate mRTECs and provided the ideal cellular resources for the basic and clinical research related to renal and cardiovascular diseases.